TREM-1 expression in H-mDCs. mDCs and H-mDCs were generated from different donors, CM was replaced on day 3 of generation with fresh medium supplemented with cytokines, and TREM-1 was analyzed at day 4 of culture. (A) TREM-1 mRNA expression. Total RNA was reverse-transcribed and tested for TREM-1 expression by quantitative RT-PCR. CAXII mRNA levels were assayed in parallel as positive control. Expression changes were evaluated as detailed in “Real-time RT-PCR.” Data are expressed as mean normalized gene expression values, calculated on the basis of triplicate measurements for each experiment/donor, relative to the values obtained for the reference genes. (B) TREM-1 protein expression. Total cell lysates were prepared from mDCs generated from 3 of the donors shown in panel A under normoxic (−) or hypoxic (+) conditions. Proteins (100 μg) were resolved on 10% SDS-PAGE, and the blots were hybridized with Abs directed to TREM-1 and β-actin as a loading control. A representative immunoblot is shown. Strong bands are seen at approximately 30 kDa, the expected TREM-1 molecular weight. (C) TREM-1 surface expression. mDCs and H-mDCs were double-stained with anti-CD83-FITC and anti-TREM-1-PE Abs and analyzed by flow cytometry on a FACScan, as specified in “Flow cytometry.” Cells were electronically gated according to their light scatter properties to exclude cell debris. Left panel: Results from one of 8 independent experiments are shown as dot plots. The percentage of single- and double-positive cells is indicated: TREM-1/CD83 double-positive cells are contained in the top right quadrant, whereas CD83 and TREM-1 single-positive cells are contained in the bottom right and top left quadrants, respectively. Cells stained with control Abs were contained the bottom left quadrants. Right panel: Data are expressed as percentage of TREM-1⁺ cells within CD83⁺ mDCs and H-mDCs generated from 8 individual donors (dots). Horizontal lines represent median values for each group. P value by the Student t test is indicated. (D) sTREM-1 secretion. Cell-free supernatants were harvested and assayed for sTREM-1 content by ELISA. Data were obtained from the same preparations analyzed in panel C and are expressed as picograms/1 × 10⁶ cells/mL (dots). Horizontal lines represent median values for each group. P value by the Student t test is indicated.